Adjuvant

Abstract

To provide a novel adjuvant that is sourced from natural substances, as well as an interferon-α production inducer or a dendritic cell activator.SOLUTION: This invention has been completed with the findings that Bacillus subtilis has superior effects in inducing interferon-α production or activating dendritic cells.SELECTED DRAWING: Figure 1

Description

[Technical field]

[0001] The present invention relates to an immunostimulant, an interferon-α production inducer or a dendritic cell activator, as well as foods, beverages, and the like containing them. [Background technology]

[0002] There is a demand for the development of foods and medicines that can normally maintain or improve immune function, which is extremely important in preventing and improving diseases.

[0003] Interferon-α (INF-α) produced by dendritic cells is a type of cytokine that is produced when the body recognizes and responds to foreign substances. The produced IFN-α is known to activate NK cells and macrophages, and is considered to be an important factor in activating natural immunity. Therefore, ingredients that enhance immunity by inducing the production of IFN-α are attracting attention.

[0004] Patent Document 1 discloses lactic acid bacteria capable of activating plasmacytoid dendritic cells (pDCs) and inducing IFN-α production, and Patent Document 2 discloses an immunostimulating composition that contains at least one active ingredient selected from the group consisting of Pediococcus acidilactici cells, bacterial components, and cultures, as well as processed products thereof, and that has an effect of activating innate immunity and adaptive immunity by plasmacytoid dendritic cells, and that has a higher effect of promoting the production of interferon-α and interleukin-12 by plasmacytoid dendritic cells than when the same amount of Pediococcus pentosaceus NRIC99 strain is used as the active ingredient. [Prior art documents] [Patent documents]

[0005] [Patent Document 1] Patent No. 6598824 [Patent Document 2] Patent No. 6796299 Summary of the Invention [Problem to be solved by the invention]

[0006] The present invention provides a novel immunostimulant, interferon-α production inducer, or dendritic cell activator derived from a natural product. [Means for solving the problem]

[0007] The inventors have discovered that Bacillus subtilis has an excellent interferon-α production inducing effect or dendritic cell activating effect, and have completed the present invention.

[0008] That is, the present invention relates to the following aspects [1] to [6]. [1] An interferon-α production inducer or dendritic cell activator containing Bacillus subtilis as an active ingredient. [2] The interferon-α production inducer or dendritic cell activator described in [1], which contains a sporulation-deficient strain of Bacillus subtilis as an active ingredient. [3] An immunostimulant for inducing interferon-α production or activating dendritic cells, containing Bacillus subtilis as an active ingredient. [4] The immunostimulant described in [3], which contains as an active ingredient a sporulation-deficient strain of Bacillus subtilis. [5] 5×10 7 In the presence of Bacillus subtilis at 1 × 10 6 The immunostimulant described in [3] or [4] contains Bacillus subtilis as an active ingredient, and has the ability to activate dendritic cells, as represented by an indicator of inducing the production of IFN-α of 20 pg / mL or more after culturing bone marrow cell-derived dendritic cells (units / mL) in a CO2 incubator at 5% CO2 and 37°C for 48 hours. [6] A food, drink, cosmetic, pharmaceutical, or feed comprising the interferon-α production inducer or dendritic cell activator described in [1] or [2], or the immunostimulant described in any of [3] to [5]. Effect of the Invention

[0009] The present invention provides a novel immunostimulant, interferon-α production inducer, or dendritic cell activator derived from a natural product. Since the effects of immunostimulation and the like are observed in killed bacteria after sterilization, the use of killed bacteria facilitates hygiene management of the production facility and quality control of the product, and allows efficient production of the immunostimulant. In addition, since the active ingredient is not a chemically synthesized product but a bacteria that has been eaten, it is ideal as an immunostimulant for which continuous long-term intake is desired. [Brief description of the drawings]

[0010] [Figure 1] 1 shows the results of evaluating the ability of a sporulation-deficient strain of Bacillus subtilis to induce INF-α production in dendritic cells at various cell concentrations. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0011] The present invention relates to an immunostimulant, an interferon-α production inducer, or a dendritic cell activator containing Bacillus subtilis as an active ingredient, which has an interferon-α production inducing effect and a dendritic cell activating effect, and is effective as an immunostimulant for inducing interferon-α production or activating dendritic cells.

[0012] The immunostimulant of the present invention contains Bacillus subtilis as an active ingredient, 7 In the presence of Bacillus subtilis at 1 × 10 6 The active ingredient is preferably Bacillus subtilis, which has the ability to activate dendritic cells, as represented by an index of inducing the production of IFN-α of 20 pg / mL or more after culturing bone marrow cell-derived dendritic cells (cells / mL) in a CO2 incubator at 5% CO2 and 37°C for 48 hours, and the amount of IFN-α produced is preferably 25 pg / mL or more, more preferably 30 pg / mL or more, 40 pg / mL or more, or even more preferably 50 pg / mL or more. 8 When Bacillus subtilis cells / mL are used, the INF-α production amount is preferably 20 pg / mL or more, 25 pg / mL or more, 30 pg / mL or more, 40 pg / mL or more, or 50 pg / mL or more, and more preferably 1×10 7When Bacillus subtilis cells / mL are used, the amount of IFN-α produced is preferably 1 pg / mL or more, 2 pg / mL or more, 3 pg / mL or more, 5 pg / mL or more, or 10 pg / mL or more. The dendritic cells to be activated are preferably plasmacytoid dendritic cells. Evaluation is performed according to the evaluation of dendritic cell activation ability using IFN-α production induction as an index, as described below.

[0013] The Bacillus subtilis described in the present invention may be live or killed as long as it is Bacillus subtilis that serves as an active ingredient of the functional preparation, and is not particularly limited, but is preferably Bacillus subtilis subsp. subtilis, and more preferably Bacillus subtilis NBRC3009, Bacillus subtilis NBRC3013, Bacillus subtilis NBRC3335, Bacillus subtilis NBRC13169, or other natto bacteria, which can be obtained from the National Institute of Technology and Evaluation, etc. In addition, if a spore-forming deficient strain is used, killed bacteria can be prepared under mild sterilization conditions of 100°C or less, which prevents contamination of manufacturing equipment due to insufficient sterilization, which is a problem with spore-forming bacteria, and makes it easy to add to various foods and to formulate them.

[0014] The Bacillus subtilis used in the present invention is not particularly limited, but is preferably a sporulation-deficient strain, and examples of methods for obtaining a sporulation-deficient strain include a method using genetic recombination and a method using mutation, but a method using natural mutation is preferred. A method for obtaining a sporulation-deficient strain by natural mutation is not particularly limited, and examples include a high-temperature culture method, a random method in which wild-type and defective strain colonies are distinguished by coloring with melanin pigment, and a method using the catabolite repression-like phenomenon (JF Michel, B. Cami, P. Schaeffer: Ann. Inst. Pasteur, 114, 11; 21 (1968)), but a method using the catabolite repression-like phenomenon is preferred. A sporulation-deficient strain obtained by a method using the catabolite repression-like phenomenon is deficient in sporulation as well as lysozyme activity and transformation ability, so it can be passaged without problems caused by bacteriolysis and can maintain the trait of sporulation deficiency.

[0015] For the cultivation of Bacillus subtilis, a conventional medium for bacterial cultivation can be used, and any synthetic or natural medium can be used as long as it contains a carbon source, a nitrogen source, inorganic substances, and other micronutrients required by bacteria. As the carbon source, glucose, sucrose, dextrin, starch, glycerin, sorbitol, citric acid, molasses, etc. can be used. As the nitrogen source, inorganic salts such as ammonium chloride, ammonium nitrate, ammonium sulfate, ammonium phosphate, amino acids such as DL-alanine, L-glutamic acid, peptone, meat extract, yeast extract, malt extract, corn steep liquor, and other nitrogen-containing natural substances can be used. As the inorganic substances, monosodium phosphate, disodium phosphate, monopotassium phosphate, dipotassium phosphate, magnesium sulfate, ferric chloride, etc. can be used.

[0016] The culture conditions can be set appropriately, but it is preferable to perform aerobically liquid culture by aeration, shaking, stirring, etc., the culture temperature can be, for example, 10 to 50°C, and preferably 20 to 40°C, the culture time can be, for example, 2 to 72 hours, and preferably 4 to 48 hours, and more preferably 6 to 36 hours, and the pH of the medium can be, for example, 4.0 to 9.0, and preferably 5.0 to 7.5.

[0017] Sterilization may be performed after the culture, and the sterilization conditions are not particularly limited as long as they are common methods, but for example, the heating temperature is 70 to 150° C., and the heating time may be determined depending on the temperature, but is usually 1 to 60 minutes. Although both live and killed bacteria have an immunostimulating effect, an INF-α production inducing effect, or a dendritic cell activating effect, it is preferable to use killed bacteria, and the use of killed bacteria facilitates hygiene management of the production equipment and quality control of the product, and allows efficient production of the immunostimulant, etc.

[0018] The bacterial cells can be collected by removing the medium using a centrifuge or the like, washing the bacterial cells with a buffer solution, physiological saline, sterile water, or the like, and then collecting the bacterial cells by solid-liquid separation using a centrifuge or the like. The bacterial cells may then be powdered by air drying, spray drying, vacuum and / or freeze drying, or the like.

[0019] The immunostimulant, INF-α production inducer or dendritic cell activator of the present invention has an active ingredient derived from a natural product and is easy to produce, and therefore can be widely used and added to various products, and may be added in a liquid, refrigerated, frozen or dry state, and can be used to prepare foods and beverages, cosmetics, medicines, feed, etc. having an immunostimulatory effect. The content of the bacterial cells in various products is not particularly limited as long as the effect is observed when ingested, but is preferably 0.01 to 20% by weight, more preferably 0.05 to 10% by weight, and even more preferably 0.1 to 5% by weight. EXAMPLES

[0020] The present invention will be described in detail below with reference to examples, but the present invention is not limited to the following examples. In the present invention, all % is by weight unless otherwise specified.

[0021] [Example 1] (Preparation of killed cell powder of sporulation-deficient strain of Bacillus subtilis) QOL Natto Bacillus subtilis (registered trademark) (manufactured by Ikeda Tohka Kogyo Co., Ltd.), a sporulation-deficient strain of Bacillus subtilis, was inoculated into a liquid medium (yeast extract: 2%, glucose: 5%, tap water: 93%) and cultured at 37°C for 24 hours with aeration and stirring, and then heat-sterilized at 90°C for 10 minutes (general viable cell count: less than 10 cells / g). The medium was then removed using a centrifuge, and the collected bacterial cells were washed with tap water and then further collected by solid-liquid separation using a centrifuge. The collected bacterial cells were dried using a spray dryer, and a killed bacterial cell powder (1.1 x 10 6 (pieces / μg) was prepared. QOL Natto Bacillus subtilis (registered trademark) is a spore-forming deficient strain that was prepared from Bacillus subtilis NBRC13169, a type of Bacillus subtilis, by natural mutation utilizing the catabolite repression-like phenomenon, and is deficient in spore formation.

[0022] (Evaluation of dendritic cell activation using IFN-α production induction as an indicator) Bone marrow cells were collected from C57BL / 6 mice (male, 8 weeks old) that had been acclimated for one week, and were cultured for 7 days in a culture medium containing Flt3-Ligand, with reference to the literature (Pierre Brawand et al., J. Immunol., 2002, 15;169(12), p.6711-6719), to induce differentiation and proliferate dendritic cells from the bone marrow cells. The obtained dendritic cells (pDC / mDC mixed cells) were cultured in a 96-well flat-bottom plate at a final concentration of 1 × 10 6 After culturing in a medium containing a sporulation-deficient strain of Bacillus subtilis at a final concentration of 1, 10, 20, 50, 100 or 1,000 μg / mL in a CO2 incubator at 5% CO2 and 37°C for 48 hours, the culture supernatant was collected and the amount of IFN-α was measured by ELISA (n=6). The results are shown in Figure 1.

[0023] As shown in Figure 1, regardless of the bacterial cell concentration, the ability to induce IFN-α production was confirmed in a test system using dendritic cells induced to differentiate from mouse bone marrow cells. Generally, IFN-α is used as an indicator of pDC (plasmacytoid dendritic cell) activation, since mDC (myeloid dendritic cells) hardly produce IFN-α. The results suggest that Bacillus subtilis has the ability to activate plasmacytoid dendritic cells, and the ingestion of killed Bacillus subtilis bacteria activates plasmacytoid dendritic cells, inducing the production of IFN-α, which is expected to have immunostimulatory effects such as antiviral effects and infection protection effects.

Claims

1. An interferon-α production inducer or dendritic cell activator, comprising Bacillus subtilis as an active ingredient.

2. The interferon-α production inducer or dendritic cell activator according to claim 1, which comprises a sporulation-deficient Bacillus subtilis as an active ingredient.

3. An immunostimulant for inducing interferon-α production or activating dendritic cells, comprising Bacillus subtilis as an active ingredient.

4. 4. The immunostimulant according to claim 3, which comprises a sporulation-deficient Bacillus subtilis as an active ingredient.

5. 5×10 ７ In the presence of Bacillus subtilis at 1 × 10 ６ Dendritic cells derived from bone marrow cells were cultured at 100 cells / mL in CO ２ Incubator with 5% CO ２ 5. The immunostimulant according to claim 4, comprising Bacillus subtilis as an active ingredient, which has the ability to activate dendritic cells as represented by an index of inducing the production of IFN-α of 20 pg / mL or more after culturing at 37° C. for 48 hours.

6. A food, drink, cosmetic, pharmaceutical or feed comprising the interferon-α production inducer or dendritic cell activator according to claim 1 or 2, or the immunostimulant according to any one of claims 3 to 5.

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